Early detection is critical to the effective treatment of many diseases, especially cancer. Research related to identifying detectable biomarkers associated with early-stage disease has indicated that nucleic acids (e.g., DNA, RNA (e.g., microRNA, mRNA, ncRNA)) provide highly specific biomarkers of cancer and other maladies. In particular, microRNAs (miRNAs) are often dysregulated in disease (see, e.g., Schwarzenbach et al (2011) “Cell-free nucleic acids as biomarkers in cancer patients” Nat. Rev. Cancer 11: 426-437; Iorio & Croce (2012) “MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics. A comprehensive review” EMBO Mol. Med. 4: 143-159). Further, miRNAs can be detected in several body fluids, including blood (Mitchell et al (2008) “Circulating microRNAs as stable blood-based markers for cancer detection” Proc. Natl. Acad. Sci. 105: 10513-10518), saliva, urine, and sputum (Iorio, supra). Thus, miRNAs provide an accessible biomarker useful for early diagnosis and treatment of diseases such as cancer.
However, despite their promise as diagnostic biomarkers, the sensitive and specific detection of miRNAs has proven challenging. For example, the low abundance of diagnostic miRNAs in a milieu of other nucleic acids hinders the reliable detection of the relevant diagnostic miRNAs. Existing assays based on polymerase chain reaction (PCR), while highly sensitive, require extraction and amplification steps that are associated with several hours of time to execute. Further, many methods based on nucleic acid amplification are known to introduce bias into results (e.g., amplification products do not accurately reflect the sequence, composition, and quantity of input samples). Other extant methods for amplification-free detection of nucleic acid targets typically utilize thermodynamic discrimination by a nucleic acid probe that hybridizes to a complementary sequence within the target (see, e.g., Tan et al (2004) “Molecular beacons” Curr. Opin. Chem. Biol. 8: 547-553; Sui et al (2011) “An ultra-sensitive DNA assay based on single-molecule detection coupled with hybridization accumulation and its application” The Analyst 136: 3950; Ostergaard & Hrdlicka (2011) “Pyrene-functionalized oligonucleotides and locked nucleic acids (LNAs): Tools for fundamental research, diagnostics, and nanotechnology” Chem Soc Rev 40: 5771-5788; Trcek et al (2012) “Single-mRNA counting using fluorescent in situ hybridization in budding yeast” Nat. Protoc. 7: 408-419; Zhang et al (2012) “Optimizing the specificity of nucleic acid hybridization” Nat. Chem. 4: 208-214). However, these existing methods face two main difficulties. First, in the absence of amplification, high-sensitivity detection generally requires single-molecule measurements that are frequently hampered by matrix-dependent background signals resulting in an incomplete discrimination of target sequences above background (see, e.g., Gunnarsson et al (2008) “Single-Molecule Detection and Mismatch Discrimination of Unlabeled DNA Targets” Nano Lett. 8: 183-188; Chan et al (2010) “Direct Quantification of Single-Molecules of MicroRNA by Total Internal Reflection Fluorescence Microscopy” Anal. Chem. 82: 6911-6918). Second, essentially all existing methods rely on thermodynamic discrimination, which places fundamental physical limits on the specificity of detection and thus results in the incomplete discrimination of target molecules relative to spurious non-target molecules (Zhang, supra). Thus, a sensitive and specific assay for the amplification-free detection of miRNAs in minimally treated native biofluids is needed to provide a rapid and reliable identification and/or quantification of miRNA biomarkers.